The mechanisms that allow cells to sense and respond to oxygen (O2) are of fundamental importance in understanding how environmental factors impact upon a myriad of physiological, pathological, and developmental processes. Recently the cellular O2-sensor/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase enzymes (PHD). In hypoxic conditions the hydroxylase activity of these enzymes are inhibited. The failure to hydroxylate hypoxia-inducible factor-l alpha leads to its accumulation. The cardiovascular system displays exquisite sensitivity to O2 with a characteristic constellation of both acute and chronic responses. Thus, the candidate proposes using the heart cell as a model system to explore the specific mechanisms that are affected by this O2-sensing pathway. New data by the candidate suggests that the PHD oxygen sensor may play a large role in the pathophysiology of the heart. Specific inhibitors of PHD activate the cellular hypoxic response, and lead to induction of two stress-response proteins; nitric oxide synthase-2 (NOS-2) and heme oxygenase-1 (HO-l). The overall goal of this proposal is to begin to explore consequences of the PHD-pathway activation and the nature of its role in the cellular response to hypoxic stress. The goal of Specific Aim 1 is to validate the use of PHD inhibitors as model agents. Neonatal myocyte cultures will be used to examine chronic PHD-pathway activation in order to evaluate if cellular responses to chronic hypoxia are recapitulated by PHD inhibitors. Specific Aim 2 capitalizes on the finding that NOS-2 protein expression is highly induced by PHD-pathway activation. The goal of this aim is to understand how PHD inhibitors lead to NOS-2 induction. NOS-2 expression will be evaluated at the transcriptional level with promoter analysis. Possible regulation of NOS-2 at the mRNA level will also be addressed by these studies. The most prominent acute response of the heart to hypoxia is a down regulation of contractility. The goal of Specific Aim 3 is to critically examine the notion that the PHD oxygen sensor plays a role in mediating the acute contractile dysfunction that is seen with ischemic or hypoxic insult.